Toxoplasma gondii is an important opportunistic pathogen that causes severe complications in immunocompromised individuals and can cause severe congenital defects in first trimester fetuses. Toxoplasma, like other apicomplexan parasites, is an obligate intracellular parasite and the tachyzoite form, which causes an acute infection, cannot survive long outside the host cell. The parasite has therefore developed regulatory mechanisms that allow it to monitor the health of a host cell, escape from it when it dies or is compromised, e.g., due to the heavy parasite load late into intracellular replication, and attach and invade into a new host cell. The activation of the egress and invasion mechanisms is controlled by the increase in parasite cytoplasmic calcium in response to host cell breakdown. This in turn activates the different elements of the egress, attachment and invasion pathway through the activation of at least one protein kinase TgCDPK1, which is required for parasite motility and host cell attachment. Parasite motility is mediated by an actomyosin-based motor and we have found that the parasites' major myosin, myosin-A, is present in a complex with three other proteins, one of which is TgMADP, the protein that anchors the myosin-A complex in the parasite pellicle. In addition, this protein appears to be involved in the regulation of parasite motility, as it is differentially phosphorylated depending on whether Toxoplasma motility is activated or not. We are proposing three specific aims for this project. First, we want to determine the manner in which TgCDPK1 controls parasite motility and host cell attachment by disrupting the TgCDPK1 gene and by characterizing and identifying the TgCDPK1 effectors in Toxoplasma. Second, we propose to continue with the structural and functional characterization of the myosin-A complex and its subunits in Toxoplasma. Finally, we are planning to determine which kinases/phosphatases control the phosphorylation state of TgMADP, which sites are phosphorylated, and the manner in which this affects the myosin-A complex and Toxoplasma motility. The long-term goal of this project is to determine and characterize in molecular detail the regulatory pathways that control the motility and host cell invasion by Toxoplasma gondii and other apicomplexan parasites. [unreadable] [unreadable]